Project summary/Abstract Increased lifespan in developed countries has led to a higher prevalence of aging-related neurodegenerative disorders such as Alzheimer Disease (AD). These diseases are characterized by the accumulation of misfolded proteins in aggregates. Under stress conditions, eukaryotic cells efficiently over-express molecular chaperones or heat shock proteins (HSPs), preventing proteins from unfolding and forming aggregates. The robust expression of HSP70 relies on the Heat Shock transcription Factor 1 (HSF1) and the translation elongation factor 1A1 (eEF1A1) which links HSP70 transcription to translation. This coupling suggests a role for the translational machinery in activating the stress response to adapt the transcriptome and assist protein quality control. We hypothesize that certain neurons are prone to accumulate unfolded proteins because of their inability to activate an efficient stress response. This deficiency could result from the switch in the expression of eEF1A1 to the isoform eEF1A2, characteristic of postnatal development of specific neurons. eEF1A2 is unable to mediate HSP70 expression and could enhance neuronal vulnerability to accumulate unfolded protein aggregates. Hence, the goals of this proposal are: 1) Engineer a ?therapeutic? eEF1A isoform to dissociate protein aggregates that cause neurodegeneration and 2) Characterize how eEF1A isoforms tune the molecular response to damage in hippocampal neurons. The stress response mechanisms as well as eEF1A protein are highly conserved between yeast and mammals and protein aggregation models mimicking human diseases already exist. Therefore, the versatile and fast manipulation of the yeast S. cerevisiae is tremendously advantageous for this proposal. For each new therapeutic eEF1A isoform, the expression of these molecular chaperones will be characterized at the single cell and single-molecule level. The mechanism and efficiency of the selected isoforms on restoring protein homeostasis in individual cells will be tested. Single cell and single-molecule techniques provide the spatial and temporal resolution required to interrogate rare cell populations like hippocampal neurons. Therefore, primary hippocampal cultures will be subjected to different stresses and single molecule FISH will be used to assess molecular chaperone biogenesis. These experiments will identify the localization of HSPs genes, their transcriptional output and the distribution of the mature transcripts. In parallel, and considering the amino acid differences between eEF1A1 and eEF1A2, a therapeutic EF1A2 isoform will be engineered to fulfill chaperone induction without altering neuronal translational competence. This proposal will stablish a new perspective on the etiology of neurodegenerative disorders and approximations to treat them. ! !